BY (From





the Western


W. H. WARD, AND H. L. FEVOLD Laboratory,* Albany, California) October 2,1944)

(Received for publication,

In 1922 Fleming (1) applied the term lysozyme to a bacteriolytic agent found in the tissues of a number of speciesof animals. The highest concentration of the agent has been found in hen’s egg white which, in a dilution of about 1:500,000, has the power to dissolve a thick suspension of test organism. Previous chemical work has been concerned only with the active principle from egg white. Available evidence, contributed mainly by Abraham (2), indicates that lysozyme is a basic protein with a molecular weight near 18,000. Abraham reported that it is stable when heated in acid solution but very heat-labile in alkali. Acetone, ether, and alcohol precipitate the active material from aqueous solution without destroying it. This latter property is the basis of the methods of preparation devised by Roberts (3) and Meyer et al. (4). In these methods the initial purification is obtained by rendering most of the egg white proteins insoluble by selective acetone denaturation at the alkaline reaction of native egg white. In the method of Meyer et al. (4) the precipitated proteins are first extracted with a mixture of alcohol and aqueous acetic acid. This step is followed by concentration of the extract under a vacuum, precipitation with alcohol, solution of the precipitate, and isoelectric precipitation of inactive protein with sulfuric acid. The lysozyme is then precipitated by flavianic acid. Repeated extraction of the dye-lysozyme complex with alcoholic ammonia liberates the active portion of the complex. The insoluble residue containing lysozyme is freed from ammonia by washing with alcohol and ether, followed by drying under a vacuum. Investigations dealing with the therapeutic usefulnessof lysozyme have been reported by Russian workers (5). They report favorable results from treatment of ulcers of the eye, postoperative infections, burns, infected wounds, and sinus infections. It appears, therefore, that lysozyme might find wide application if sufficient material could be made available in purified form. The present communication reports the results of work which was initiated for the purpose of developing a method for the preparation of large quantities of lysozyme. The method reported here makes possible the recovery of lysozyme from egg white in high yield, and the
* Bureau ministration, of Agricultural and Industrial Chemistry, United States Department of Agriculture. 43 Agricultural Research Ad-

Downloaded from by guest, on February 13, 2011

As routinely carried out in our work the method was reliable to approximately 15 per cent. Under standardized conditions a given change in galvanometer reading over a certain time interval corresponds to a definite amount of lytic agent. At pH values ranging from 5. attempted to make use of the ready adsorbability of lysozyme on clay. to effect elution from kaolin or charcoal. Attempts to separate the positively charged lysozyme from the negatively charged clay particles by cataphoresis were unsuccessful. obtained in apparently pure crystalline EXPERIMENTAL In addition. lysozyme was adsorbed on kaolin suspended in buffer solutions.l Puri$cation of Lysoxyme by Adsorption and Elution Methods-Fleming (1) reported that lysozyme is readily adsorbed on charcoal. By this method the rate of lysis of a suspension of killed Micrococcus Zysodeikticus organisms is measured as indicated by the rate of change of light transmission recorded by a photoelectric calorimeter. Treatment of the kaolin-lysozyme complex with various buffer solutions ranging from pH 4. A lysozyme unit is defined as the amount of lysozyme in 1 ml. on February 13.jbc. but with indifferent success. including phosphates.5 to 9. By repeated solution and reprecipitation of euglobulin fractions containing adsorbed lysozyme. kaolin. He was unable. acetates.5 to 14 did not elute the active material. and subsequent studies have shown that lysozyme is adsorbed tenaciously on many substances. dialysis against buffers of different strength and hydrogen ion concentration did not prevent adsorption. and porcelain. preparations with an activity 25 to 30 times that of egg white. Likewise. silicic acid. However. and filter paper adsorb the active material. form. cellulose. In studies on the purification of lysozyme. comparison of its activity with that of crystalline lysozyme obtained later showed that it was of high purity. of fresh egg white. Among these were bentonite (a very finely divided mont1 While this preparation was not investigated further. and dilute ammonia by guest. of a standard sample of liquid egg white. Downloaded from www. however. were obtained in low yie1d. so the unit might be stated to be the amount present in 1 ml. glycocholates. The method of Boasson (6) was used for assay purposes. We investigated the capacity of several adsorbing materials to bind lysozyme. 2011 . the amount of lysozyme in fresh egg white has been found to be very constant.44 ISOLATION OF LYSOZYME product appears to be essentially pure. the Russian investigators. Buyanovskaya and Jermol’eva (5). The effect of pH on the ammonium sulfate precipitation was investigated but failed to reveal conditions that would prevent adsorption of lysozyme. irrespective of pH. the product has been Puri$cation of Lysoxyme by Salt Fractionation-Attempts were made to purify lysozyme by fractionating the egg white with ammonium sulfate.0. on the dry weight basis. Wolff (7) found that norit. The characteristic of lysozyme that militated against salt fractionation was its adsorption in varying degrees on inactive protein precipitates.

WARD. Potassium chloride solution and 6 M urea solution likewise did not remove any active material. AND FEVOLD 45 morillonite clay).org by guest. Since organic bases are known to be strongly adsorbed by montmorillonite clays. When the pH is lowered. Of the substances investigated. and falls off to zero at pH 3. of egg white. of the material removed the active substance from 80 to 100 cc. was 6. The eluates were removed from the clay and assayed for lytic activity. on February 13. 10 10 10 15 12.jbc. Appreciable amounts of inactive protein are eluted by pyridine or pyridine-sulfuric acid solutions at pH values above 6. .ALDERTON. In these experiments acetic acid was replaced by sulfuric acid in order to extend the pH range.0. Investigation of the effect of pH on the elution of lysozyme by pyridine solutions proved that it could be eluted very efficiently at the proper pH. 1 gm.5 15 15 15 adsorbed per cent 96 500 1850 1200 2000 2000 3000 1900 2000 480 1600 1190 1980 1700 2900 1850 1950 87 99 99 85 97 97 97 cent aqueous acetic acid did elute about 30 per cent of the adsorbed activThe pH of this solution. Rliquots of a suspension of clay on which lysozyme had been adsorbed were stirred with pyridine-sulfuric acid solutions at various pH values. thereby effecting a marked purification of the adsorbed bactericide. Panther Creek bentonite. solutions of o-phenylenediamine and p-phenylenediamine were tried but the amines were adsorbed on the clay without eluting lysozyme.0.2. Supersorb (a synthetic zeolite). removal of the active material begins at pH 6. a mixture of 20 per cent pyridine in 2 per TABLE Adsorption 1>ysozyme maz z&nils Downloaded from www. 1). bentonite was found to be the most efficient adsorbent for lysozyme from native egg white.2. The elution of lysozyme was found to be specific with regard to the pH of the eluting solution (Fig. rises sharply to a maximum at pH 5. Table I gives the data from a number of representative experiments. However.0. as determined by a glass electrode. We have confirmed the results of previous workers in that we were unable to elute the lysozyme with aqueous inorganic buffer solutions. 2011 I by Bentonite Lysozyme unils of Lysozyme Bentonite per liter egg white gm. ity. and Duolite (a synthetic resinous cation exchanger).

The clay is next washed three times with 300 ml. of a 5 per cent aqueous solution of pyridine. Elution of lysozyme is then accomplished by washing the clay by guest. 1. which has been adjusted to pH 5. Drying the solution in the frozen state gives a white solid. For best results elution should follow adsorption within 24 hours. The activity of the material is constant from one preparation to another within the limits of error of the assay method. of the .5 M phosphate buffer at pH 7. To each liter of egg white are added 150 ml. The precipitate readily dissolves in distilled water and the solution may be freed from salt by dialysis . of a 10 per cent suspension of bentonite in 1 per cent KC1 and the mixture is stirred vigorously (foaming being avoided) for 3 to 5 minutes. Typical data on distribution of protein solids. The clay is separated from the suspension in a Sharples centrifuge and washed with 0. the following method was devised for separating lysozyme from other egg white proteins. on February 13. each time with 300 ml. (total 900 ml.6 M precipitates the active substance. Removal of the eluates Downloaded from www.46 ISOLATION OF LYSOZYME Based on the facts reported above. 2011 pft Meter Readings FIG.5 to remove egg white mechanically held in the clay mass. Effect of pH on elution of lysozyme from bentonite from the clay is accomplished by centrifuging in a batch type centrifuge. Addition of ammonium sulfate to a concentration of 2. yield of lysozyme. and activity of the preparations obtained are given in Table II.0 may be accomplished in a number of ways.0 (glass electrode) by the addition of sulfuric acid.jbc. which may be collected by centrifugation or filtration. Concentration of the active material contained in the eluates at pH 5. These data show that as high as 90 per cent of the lysozyme present in egg white can be obtained by this process in a concentration of 35 to 40 times that of the original egg white solids.) of a 5 per cent aqueous solution of pyridine to remove inactive adsorbed proteins.

0046 0.0047 0. <2 10 <2 93 93 89 95 10 285 320 330 300 * Egg white units per gm. 2011 II from Bentonite Eluted protein solids per unit sdsorbed lysozyme b-m. 1 2 3 4 1 2 3 4 1 2 3 4 of protein 0. pH 7-r Alkaline 7-8 pyridine eluates. preferably in the cold. El&ion of pozyme Exp$mnt Clag* Elution Units per gm. 0. after being dried from the frozen state. is a light. Concentration can be effected also by precipitation with 75 per cent alcohol or acetone. two 1 per cent solutions of the TABLE ~. the activity of each being essentially that of the starting material within the limits of the determ. Fractionation of Purified Lysozyme with Ammonium Sulfate-h an attempt to purify the active material further.0055 by guest.inations. The dialysis is then completed against running distilled water for 24 hours. followed by dialysis. The least and t. No significant concentration of activity was observed in any of the fractions. protein solids per cent Phosphate buffer eluates.0033 0.0017 0.Eluant Downloaded from www.0018 0. It appears from these experiments that the preparations obtained on elution were grossly homogeneous. AND FEVOLD 47 activity indicated.0027 0. on February 13.0027 0.0014 0. contains of protein. .0020 0. adjusted to pII 5 and 7. fluffy. The solute.0032 solids per ml.he most soluble fractions appear to have less activity than the rest of the material but the amounts of solids in these fractions were so small that the accuracy of the analysis is questionable. and precipitates were removed at increments of 0. and 8 to 10 lysozyme eluted lysozyme in distilled water.ALDERTON. were fractionated with ammonium sulfate.1 to 0. The salt was added by dialysis through a rotating membrane (8) at I”.2 M ammonium sulfate concentration and the solids and activity determined on the precipitates and filtrates (Table III). pH Pyridine-sulfuric PH 5 acid eluates. The active eluates are placed in cellophane bags and dialyzed against running tap water until no odor of pyridine remains.0033 0.12 gm.jbc. stable powder. WARD. We have found it more convenient to prepare the dry lysozyme powder in the following manner. which remains stable at room temperatures for several months.

but since lysozyme does not pass through the membranes it appears permissible to ascribe the activity measured before dialysis to the undialyzable fraction. it should be possible to remove digestible contaminating proteins by enzyme degradation followed by dialysis.78 0.18 100 338 304 306 311 112 material took place on dialysis after digestion except in the case of the pepsin-treated material. were digested for 24 hours at 30” with bacterial proteinase (pH 7. Filtrate Ppt.93 318 0.8 1. papain activated with cysteine (pH S. Filtrate material 45 980 316 505 245 246 125 141 83 20 1090 10 930 223 586 305 358 160 147 122 20 0. .4 2. Filtrate Ppt. Filtrate Ppt.* No significant loss of activity took place on digestion with any of the enzymes. Two conclusions may be drawn from these experiments.0 2.05 3.06 0. Accordingly.20 . mold proteinase (pH 7.81 2.28 0.4 1.jbc. 10 ml. other than pepsin. with the exception of pepsin.6 1.04 1. pepsin-digestible protein might be regarded as 2 Unfortunately the activity after dialysis was not determined. on February by guest. The activity per unit of undigestible material was then calculated.41 0. Similarly no marked loss of TABLE Downloaded from www.0 2.0). tration -- pH 5 -__ PH ‘i .00 1.40 0.O).6 1.4 1.4).48 ISOLATION OF LYSOZYME Action of Enzymes on Lysoxyme Preparation-It has been reported that lysozyme is not destroyed by proteolytic enzymes (5). only pepsin digests lysozyme at a rate measurable under the conditions of the experiment. After the activity of each digest had been determined. units at pH 6 and 7 Protein solids. 2011 III -(NH&SC concenI4 Ammonium Sulfate Fraction Fractionation of Lysozyme Assay. portions of a lysozyme solution containing 8 units per ml.48 250 308 255 310 314 265 305 352 296 200 281 275 293 293 0.43 3. The calculated activity of the undigested material was in all cases not significantly different from the determined activity before digestion (Table IV). Only small amounts of inactive.8 2.24 1. Since it is non-dialyzable. 0.63 0.18 1.4).39 0. Of the enzymes used. each was dialyzed against distilled water for 16 hours and the solids determined.4). trypsin (pH 7.18 3. gm. and pepsin (pH 2.53 0. and no proteins that are digested by the enzymes used. were present in measurable quantities. Filtrate Ppt.31 0.4 Starting Ppt.--~PH 5 PH 7 PH 5 I___ PH 7 1.

5 at 96”.22 0. A lysozyme solution similar to that used for the preceding experiment was heated until a destruction of 40 per cent had taken by guest.8 was dissolved in distilled water and heated on a steam bath at a temperature of 96”.24 0. and samples were removed for analysis at regular intervals. TABLE IV Hydrolysis Enzyme of Lysozyme Preparation Assay total units by Proteolytic Solids Enzymes of Activity of undigested material Destruction activity Downloaded from www. The solution . Lysozyme powder at pH 2. WARD. we determined the effect of heating alone on the activity of lysozyme.5 50 .*I 46 44 40 30 20. While the rate of destruction was slow.0) 50. Since many proteins are sensitized to enzyme action by heat. since no increase in activity of the undigested material was apparent.5 6. Starting material Bacterial proteinase Mold proteinase Trypsin Pepsin Papain 80 72 72 79 35 74 10 1 56 7 Stability Tiie nin. 2011 gm. 0 Assay units TABLE V of Lysozyme to Heat* LOSS LOSS units 0 per cent 0 9 13 21 40 60 5 10 15 25 50 80 * Heated in HCI (pH 3. AND FEVOLD 49 having been present. only if the rate of digestion of the inactive material is greater than that for lysozyme.jbc. Before proceeding with the digestion experiments. 4. This conclusion is valid. experiments were carried out to determine whether the protein or proteins in the lysozyme preparation could be thus sensitized and a differential digestion then be obtained.5 10. 0.22 0. however.ALDERTON.26 0. a progressive decrease in activity was found which amounted to a 60 per cent loss after 80 minutes of heating (Table V). on February 13.5 20.5 30 Lysozyme has been reported to be stable to heat in acid solution but very labile at alkaline reactions.25 per cent 10 &ts per 305 327 327 322 305 302 gnc.12 0.

the activity of the undigested remainder was the same as that of the original unheated material with one exception. remained insolution.8. whereas. and someprecipitation of the solute took place. per ml. per ml.5 0. No difference between the lytic activity .3 17. on February 13. w&its L!*.5.1 0.0 at the beginning. Electrodialysis of Lysozyme Solutions-Solutions of lysozyme which had been dialyzed against distilled water were further freed from electrolytes by electrodialysis. The results are presented in Table VI. In no case was there any indication of a differential digestion resulting in increased activity of the undigested active preparation.6 9. It is also evident from these experiments that some change in the lysozyme molecule is induced by heating which does TABLE VI Hydrolysis Enzyme Downloaded from www. 6 mg. that is. had risen to approximately 9. when papain was used. With adjustment of the electrodialyzed solution to pH 10 to 10. The pH.50 ISOLATION OF LYSOZYME was then treated with the various enzymes in the same manner as described for the unheated material. which may mean that digestion of the material. per cent Starting material Heated material Bacterial proteinase Mold proteinase Trypsin Pepsin Papain 46 27 22.08 by guest. at pH 9 and 11. or until no appreciable change in conductivity of the solution took place. readily dissolved. Apparently the rate of digestion by all the enzymes had been markedly accelerated.06 0.5. was not complete or the products of digestion were not completely dialyzable. t Calculated as per cent of activity after heating. which was approximately 7. The electrodialysis was carried out at 1’ for approximately 18 hours.2 16.5. After removal of the digested part by dialysis.03 0. At pH 10. heavy precipitation resulted.16 0.07 293 41 (By heat) 18 35 66 99 39 171 280 293 303 * Heated in HCl (pH 3) at 98” for 90 minutes. 2011 of Heat-Treated Lysozyme Assay Preparations Solids by Proteolytic Enzymic destruction activityt of Enzymes* Activity of undigested material units per gm. 150 mg. inactivated by heat. which appeared to be the point of minimum solubility. however. In this case the unit activity was reduced.jbc.02 0.16 0. At pH 11 resolution of the precipitate was apparent. and was complete at approximately pH 11.5 at the end of the experiment. not disrupt the structure necessary for lytic activity but which does sensitize the molecule to enzyme action.

hod within the pH range of 4. Therefore. and MacInnes (9). above pH 7.8 and at an ionic strength of 0. The results of all analyses are given in Table VII. The mobilities of lysozyme approximate the values reported for the globulin component of egg white designated G1 by Longsworth. Electrophoretic Behavior and Isoelectric Point of Lysozyme-Several purified lysozyme preparations have been analyzed electrophoretically by the Tiselius met. on February 13. although differing in mobility from the lysozyme preparation reported by D.ein contained in our lysozyme preparations. while in one or two trace components could be detected. AND FEVOLD 51 of the precipitates and that of the material remaining in solution in the isoelectric region could be demonstrated. This component was designated by them as G1. Phosphates depress the mobilities of lysozyme and G1 from the values in buffers containing only monovalent ions to approximately the same extent. except for the statement that an isoelectric point was not Downloaded from by guest. cm. The descending boundary in many of the analyses spreads considerably. sometimes with a small spike.0. 2011 .5 per cent of the proteins present in egg white.8 per cent of the egg white proteins. The average amount of this component comprised 2.ALDERTON. Cannan. In the acid buffers the mobilities were lower than reported for G1.8.8.5 to 11. whereas the average yield obtained in our preparations was 2. two different samples were found to have mobilities closely similar to that reported for G1. which is in close agreement with the isoelectric point of the prot. Longsworth. 2).0. no evidence for the presence of more than one component was found. extrapolation of the mobility curve shows an isoelectric region around pH 10. Cannan. Moore in an article by Meyer (+6. In most of the analyses the ascending boundary appeared homogeneous. beginning abruptly at the leading edge. but in Analyses 1 and 2 of Table VII the sharp leading edge coincides with this reported mobility. In all of these analyses one component comprising more than 95 per cent of the total gradient was present and in several the mat. While the mobility of G1 was not determined by Longsworth et al.jbc.erial appeared quite homogeneous. It was in these spreading boundaries that evidence of the presence of trace components was chiefly observed (see foot-note to Table VII and Fig. per volt second at pH 7. as shown in Fig. In the media near pH 7.1. These facts point strongly to the identity of G1 and lysozyme.5 to 11. The properties of G1 at higher pH were not discussed by Longsworth and his associates. The conclusion seems justified that the lysozyme preparations are essentially electrophoretically homogeneous and may consist of quite pure protein. and MacInnes (9) have reported that electrophoretic analysis of egg white proteins reveals the presence of one and only one component with an isoelectric point above pH 7.80) (10). WARD.75 X 10-j sq. 3.

01 0. V = diethylbarbiturate (barbital. The rising boundary was sharp in these cases.3 composition* 1 1 1 4 5 6 7 8 9 10 11 - 4. 52 .8$ +3.80 HA NaA HA NaA HC NaC NaCl NaHzPOd Na2HP04 HV NaV NaCl HV NaV NaCl NH.5 6.09 0.0 10.7 11.15** -2.02 0.t - * Acids and salts present in buffers used were as follows: A = acetate.13 +7. referred to 0’ by multiplying the mobility at the temperature of observation by the viscosity of water at that temperature relative to. 2011 +5.01 0. C = dimethylarsonate (cacodylate).01 0.30 +3. 7 Nearly saturated solution. $ In several analyses the falling boundary spreads considerably.3 _( Electrophoretic TABLE VII Analyses of Lysozyme Preparations Buffer nolarity -Mobility Principal of falling boundariest TIXW :oncentra.042 0.jbc.6.09 0.$ +6.02 0.33 +6. cm.3**q -3.49 +3.02 0.1 0.1.73 7.47 5.15 0.9.09 0.76 9. t Mobilities are reported in terms of 10-G sq.04 .03 6. but the total displacement during the time available for analysis was insufficient to allow as conclusive a test as in other instances. ** Apparently homogeneous.09 0.08 0. E = ethanolamine. tion.017 0. $ The sample. so that the spreading may be attributed to imperfect ionic adjustment of solution and buffer.27$ -l-3. +1.09 +6.019 0.01 0. it Analysis of the solution for biological activity and nitrogen content indicated that the lysozyme was stable under the conditions of the experiment.01 0.2 +5.1 0.6 +4. G = glycine.711 +3. 11Mobility of minor component estimated from rising side. isoelectrically precipitated. per cent protein PH BUff.01 0. veronal). sometimes with a small spike.9t +5.01 0. but in Analysis 6 showed trace by guest.01 0.5 7.6 +4.7 10.8 +4. as noted.09 0.09 0. per volt second.02 0.n +1.the viscosity of water at 0”.1 0. was apparently quite homogeneous in this medium.6$ Downloaded from www.OH NH&l NaCl G NaOH NaCl E HCl NaCl E HCl NaCl NaOH NaCl - 0.0 10. and the spike may not represent an extra component. on February 13.01 0.01 0. beginning abruptly at the leading edge.# +3.

compared remained constant. Table VII. (a) Analysis 1.027 ampere per sq. Table VII.5. . WARD. I 7 (Longsworth) Observed \ \ \ x Nod- Observed (leading edge) I 8 I 9 I IO I II I 12 I 4 I 5 I 6 PH FIG.Gacody’ote \xe Phosphate x + 32l- \ Veronol \ \ \ NH. on February 13. Svensson-Philpot patterns of lysozyme preparations analyzed at 0. after 3000 seconds. Lysozyme. Extension of the published graph to the mobility value at this pH indicates an isoelectric point near pH 10. (b) Analysis 2. \ \ - -2 - + x @ G.09 N NaCl (pH 11. k4u g i g =0 i-1 H E-3&4d a 5. mobility of lysozyme as a function of pH. by guest. after 14. AND FEVOLD 53 We found that lysozyme moved toward the anode in a medium observed. 2.000 seconds.01 N NaOH and 0.ALDERTON. after 12. Table VII.Q \ \ \ \ x Glycine-NaOH x Ethanolamine \ \ x Ethanolamine \ .80) in which its biological activity Downloaded from www.jbc. . of 0. The electrophoretic with that of the globulin G1. 2011 FIG. However. 3. Reported Lysozyme. (c) Analysis 5.000 seconds.

500 is obtained.70 to 0. Sedimentation. Crystallization has been successful under a wide range of pH values. personal communication. B. calculated from the maximum scale line displacement and area. Downloaded from www. ranging from the isoelectric region (pH 10. The measurements made at two concentrations of lysozyme gave the value of the ratio of pressure to concentration equal to 151. Since partial specific volumes were not determined and osmotic pressure measurements were made with a’slightly permeable membrane.000 to 17. 2011 . just as the deviation with phosphates suggests phosphate binding.5 and 11. The osmotic pressures were determined by extrapolating to zero time measurements with cellcphane membranes (No. and by the method of successive analysis (( 11) Equation 50) is 11. a Lamm-Polson diffusion cell (ll).5.15 M sodium chloride. The isoelectric point in these media seems to be near pH 11. Crystallization of Lysozyme-Lysozyme has been obtained in crystalline form. This circumstance suggests complex formation with the amino constituent of the medium. but if we assume values from 0. Two factors contribute to the ease of crystallization of lysozyme. Partial specific volume measurements have not been made. is regularly toward the cathode and faster than indicated by this extension of the trend of values at lower pH. in 0.5 per cent. values at A!!&. However. The pressures obtained were extrapolated to zero concentration of lysozyme by a plot of the ratio of pressure to concentration versus concentration. 300)) through which lysozyme diffuses slowly.0.5 to 0. they are in agreement with each other and also with the molecular weight of 18.000 from the sedimentation and diffusion constants obtained for this material.jbc. 3 Davis.54 ISOLATION OF LYSOZYME the mobility of lysozyme in buffers containing ammonia. or ethanolamine. From this value a molecular weight of 17. Diffusion. 13). and in a modified Hepp osmometer (12.000 given by Abraham (2). glycine. From the sedimentation rates measured at lysozyme concentrations of from 1. W were found to be about 1.9 Svedberg units. the values for the molecular weight of lysozyme must be considered as approximate. and the solubility of the amorphous material is 3 or 4 times as high as that of the crystalline form.. by guest. The correspondence of the electrophoretic and chemical properties of the highly purified lysozyme with those of the globulin G1 indicates that they are very similar and in fact may be identical.8) to 3. on February 13. It has a relatively high positive temperature coefficient of solubility. and Osmotic Pressure Measurements with Lysoxyme-Solutions of the purified lysozyme were studied in an air-driven ultracentrifuge.2 X lo-’ at 1 per cent lysozyme concentration. D.75 we can calculate molecular weights from 14.3 The measurements indicated that the lysozyme preparation is homogeneous and that lysozyme falls among the lowest weight proteins listed by Svedberg (14). W. The average diffusion constant. between pH 9.

or at pH 7.5 was carried out in the following manner. 4. Crystallization from ammonium sulfate solutions (0. X). 4 gm. containing 5 per cent of sodium chloride. of 0. and as a result of the lower solubility of the crystalline Downloaded from by guest.0 with acetate. 4. when well defined crystals were deposited as shown in Fig. which had been dissolved in dilute acetic acid at pH 6 and dried in the frozen state. Crystalline lysozyme (120 pH 4. Crystallized from 0. When the process was continued long enough. product. were dissolved in 60 CC.5. of the original material remained in solution after crystallization for 16 hours. Only 15 mg.5 with sulfuric acid. Crystallization was also effected at room temperature from 1. The rate of crystallization may be markedly increased by lowering the temperature from 21” to 4”. WARD. on February 13. per ml. The activities of the various crystalline preparations were in all casessimilar to those of the amorphous material. 2011 FIG. all of the excess amorphous solid was changed to the crystalline state. AND FEVOLD 55 Crystallization at pH 4. it seemsthat the crystal form of its salts with various .4 M) buffered at pH 5. The crystals so formed were very thin rectangular plates.jbc.4 M ammonium sulfate solutions acidified to pH 3. The solution was allowed to stand at room temperature. The amorphous material partially dissolved.2 M acetate buffer.ALDERTON. containing 5 per cent NaCl. crystals were deposited.5. Since lysozyme is a very basic protein.2 M acetate buffer at pH 4. Crystallization at the isoelectric region (pH 10 to 11) was accomplished by agitating an excess of the isoelectric protein with saturated sodium chloride adjusted to pH 11 with XaOH. resulted when the temperature was lowered from 21” to 4”. The crystalline forms obtained by the procedures outlined above seem to vary with the pH at which crystallization is carried out and with the acid used in effecting solution of the isoelectric material. of isoelectric protein.0 with phosphate.

since percentage yield and activity of egg white are not given. (4) and to behave as a homogeneous protein in the ultracentrifuge. avidin.jbc. The amounts present. with the pure lysozyme preparations we have been unable to obtain any indication that avidin and biotin are concerned in the lytic activity of Downloaded from www. Abraham and Robinson reported in a short note in 1937 (15) that crystalline material was obtained from a lysozyme preparation which was prepared by Robert’s method (3).05 N acetic acid solution in vacua over aqueous KOH. however. The quantitative yield of active material is not given. and hence found it necessary to use dead organisms in order to carry out quantitative work. the It isoelectric region. is being further DISCUSSION investigated and the results It is difficult if not impossible to compare the activity of our preparations Meyer et al. Crystallization was induced by evaporation of a 0. Robert’s unit is similar to that of Meyer et al. The fact that we have been able to obtain no evidence for the presence of two substances of varying activities and the ease of crystallization of our purified materials indica. but Abraham (2) was able to separate two fractions of different solubilities and activities. The facts presented in this paper indicate that lysozyme and the globulin called G1 by Longsworth et al. Also.16) appeared dealing with the relationship of biotin. and the mobilities are all in approximate agreement. and lysozyme. The unit is defined as the smallest amount causing complete lysis of Micrococcus lysodeilcticus in a serial dilution test. 2011 .org by guest. and the activities are likewise incapable of translation. This preparation appeared to be homogeneous in the ultracentrifuge. In the preparations used in the work reported in these papers it seems that avidin activity and lytic activity were correlated. Abraham (2) reported that he had not been able to obtain sufficient crystalline material for chemical examination. in our preliminary work with assay methods we found that the susceptibility of Micrococcus lysodeikticus varied many. on February 13. preparations contain from 2 to 6000 units per mg.56 acids may vary. (4) stated that their with those reported by other investigators. While this paper was in preparation.fold from day to day and from culture to culture.te that the two preparations may not be identical. will be reported ISOLATION OF LYSOZYME This matter later. two papers (10. and it was intimated that lytic activity may In preliminary experiments depend on the presence of avidin and biotin. Abraham (2) reported Robert’s preparation (3) to be an improvement over that of Meyer et al.. 2 years later. appears unlikely from available information that more than one substance with these characteristics is present in egg white in the amounts found for G1 and lysozyme. are identical.

Lysozyme has been prepared in crystalline form. Brown. Boston.5 to 5. 622 (1939). has been developed. diffusion. The eluate is dialyzed and dried in the frozen state.. and by electrophoretic and sedimentation studies.000). Jones of the Western Regional Research Laboratory for the preparation of the Micrococcus lysodeikticus organisms for test purposes and the preparation of the photomicrograph respectively. Quart. 2. We are also indebted to E. 2. Crystallization has 5. 33. Cambridge. 3. Abraham.0. H. 89 (1937). These communication. P. The purified substance is stable in acidified solutions and relatively stable also in alkaline solutions. Roberts. depending on the pH of crystallization and the acid used in dissolving the protein. It is isoelectric at some point between pH 10.0.0). Fleming. Proc. Roy. . 306 (1922). J. It seems probable that lysozyme and the substance termed G1 by Longsworth et al. At pH 11. Lysozyme is a basic protein of low molecular weight (about 17. Massachusetts Institute of Technology. A. AND FEVOLD 57 more fully in a later lysoeyme. been effected at the isoelectric region (pH 10. WARD. Laboratory of Physical Chemistry. E. Ezp.. The lysozyme preparations have been shown to be essentially pure by salt fractionation. 27. by their behavior toward enzymes. are identical. for carrying out the sedimentation. Department of Physical Chemistry. Biochem. Series B.8).ALDERTON. experiments will be reported SUMMARY 1. and osmotic pressure measurements reported in this communication. (6) elution of inactive contaminating proteins from the clay by successive washings with phosphate buffer (pH 7 to 8) and 5 per cent aqueous pyridine..5 no loss of activity could be detected over a period of 5 to 6 hours. Wolford and F. The crystal form appears to vary. Sot. at pH 7. Physiol. Massachusetts. 2011 1.. on February 13. Massachusetts. J. 4. We are very grateful to Dr. Harvard Medical School. A. Oncley. and (c) elution of the active material with pyridine-sulfuric acid solution at pH 5. London.. and Dr. BIBLIOGRAPHY Downloaded from www. A white powder is obtained containing 85 to 90 per cent of the lysozyme contained in the egg white. L. A method for the isolation of lysozyme from egg white.jbc. 93. and in acid solutions (pH 3. 3. on the (a) adsorption of Iysozyme on bentonite (a montmorillonite clay). 6. in high yield The method depends and in essentially pure form. H. by guest. J.5 and 11. E.

Chem. SC. 12.. O.. Thompson. 14. G... L. K. and MacInnes. Peters.. 248 (1938). and Khorazo. 23. Immunol. and Robinson. Wolff. J. W. Chem. K. 391 (1944). 34. 88 (1927). J. 15. 8...jbc.... Meyer. Longsworth. 62. 392 (1944). O. Palmer. 140.. K. R. 2011 . Lamm. J. 2. J... Am. 9.58 ISOLATION OF LYSOZYME 4. 2884 (1939). hTo. Z.. Sot. 99. 61. 2580 (1940). Biol. 281 (1938)... O. sot. W. 13. 11. Am. Ups&en&. L. J... T.177 (1939). ezp. 10. D. I. A.. E. Cannan. Gen. H. 1. G... The ultracentrifuge. Science. Therap. Oxford (1940).. Nova acta reg. Meyer. E. D. 99. R. Physiol. Downloaded from www. 709 (1936). R. 7. 10. by guest. and Pedersen... Acta med. 303 (1936). Buyanovskaya. ges.. and Jermol’eva. U. ezp. 6 (1937).1mmunitiitsforsch. science. 16. 2.. P. E. Boasson. R. T. 50. Laurence. L. 6. Svedberg.24 (1937). u.. S. Sot. S. 113.. 5. J. K. 99. McMeekin. K. on February 13... Chem. and Saslow. L. Med. Hepp. Abraham.

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